摘要目的 观察KRAB-相关蛋白1(KAP-1)对胰腺癌Capan-2细胞株化疗耐药的影响.方法 PCR扩增KAP-1序列,克隆至pGC-FU-3 FLAG-IRES-Puromycin慢病毒表达载体；双酶切及测序鉴定正确后进行慢病毒包装并进行滴度检测.构建成功后感染人胰腺癌细胞株Capan-2细胞,48 h后使用实时定量逆转录聚合酶链反应(RT-qPCR)以及Westem blot法检测KAP-1的表达量及其相关上皮-间充质转化(EMT)标志蛋白——波形蛋白的表达.采用细胞计数试剂盒(CCK-8)方法测定不同化疗药物在不同浓度下过表达KAP-1的Capan-2细胞生长及化疗药物对胰腺癌细胞的生长抑制作用.结果 构建的慢病毒载体感染细胞48 h后,在倒置荧光显微镜下观察可见绿色荧光,病毒滴度为2×108 TU/ml.RT-qPCR检测结果表明,实验组、阴性对照组、空白对照组细胞中KAP-1的mRNA相对表达量分别为9.48±0.73、0.03±0.00、0.02±0.00,差异有统计学意义(P＜0.05)；波形蛋白的mRNA相对表达量分别为7.84±0.21、0.63 ±0.07、0.59±0.12,差异有统计学意义(P＜0.05).Western blot检测结果表明:实验组、阴性对照组、空白对照组细胞中KAP-1的蛋白相对表达量分别为2.73±0.97、0.63±0.36、0.51±0.14,差异有统计学意义(P＜0.05)；波形蛋白的相对表达量为4.75±0.53、1.00±0.26、1.25±0.91,差异有统计学意义(P＜0.05).转染过表达KAP-1慢病毒载体的Capan-2细胞株在吉西他滨、5-氟尿嘧啶、顺铂作用下的50％生长抑制所需药物浓度(GI50)值分别为27.53、3.96、3.18；阴性对照组及空白对照组分别为0.28、0.15、0.39；0.14、0.21、0.27,差异有统计学意义(P＜0.05).结论 构建了高效稳定表达KAP-1的慢病毒转染系统.KAP-1转录因子可以减慢人类胰腺癌细胞Capan-2细胞株在化疗药物的作用下的增殖速度,并降低该细胞对化疗药物的敏感性.其机制可能与过表达KAP-1影响波形蛋白上调有关.

abstractsObjective To investigate whether KRAB associated protein 1 (KAP-1) could induce drug resistance in pancreatic cancer cell lines.Methods KAP-1 amplified by polymerase chain reaction (PCR) was inserted into pGC-FU-3FLAG-IRES-Puromyc in vector,and then identified by restriction endonuclease digestion and nucleotide sequencing.The lentiviral vector pGC-FU-kap-1 was co-transfected with pHelper 1.0 and pHelper 2.0 packaging plasmids into HEK 293T cells,and the lentivirus was collected and virus titer was measured.The expression of KAP-1 and vimentin was detected by using real-time reverse transcriptase-PCR (RT-qPCR) and Western blotting when human pancreatic cancer cell line Capan-2 was infected by the lentivirus.Cell counting kit-8 (CCK-8) method was used to detect the proliferation of Capan-2 cells in the presence of drugs.Results After infected by the lentivirus,the relative mRNA expression of KAP-1 and vimentin was 9.48 ±0.73 and 7.84 ±0.21 in the experimental group,0.03 ±0.00 and 0.63 ±0.07,and 0.02 ±0.00 and 0.59 ±0.12 in the negative control group and blank control group,respectively (P ＜0.05).The results of Western blotting showed that the relative protein expression levels of KAP-1 and vimentin were 2.73 ± 0.97 and 4.75 ± 0.53 in the experimental group,0.63 ± 0.36 and 0.51 ± 0.14,and 1.00 ±0.26 and 1.25 ±0.91 in the negative control group and blank control group respectively (P ＜0.05).Half maximal inhibitory concentration (GI50) values for Gemcitabine,5-Fu and Cisplatin were 27.53,3.96 and 3.18 in the experimental group,0.28,0.15 and 0.39,and 0.14,0.21 and 0.27 in the negative control group and blank control group respectively (P ＜ 0.05).Conclusion The lentiviral vector expressing KAP-1 was constructed successfully.Overexpression of KAP-1 can inhibit the proliferation of Capan-2-KAP-1,and it can also decrease the sensitivity of the cells to drugs.The mechanism is probably related to the up-regulated expression of epithelial-mesenchymal transition marker vimrntin.